The Chagas' disease parasite Trypanosoma cruzi has the ability to infect many nucleated cell types, but its persistence in cardiac and smooth muscle is critical for disease progression. Like all obligate intracellular pathogens, T. cruzi mus couple its metabolic requirements to its host in order to survive. If the biochemical and cellular pathways that tether T. cruzi to its host cell were known - for cardiomyocytes in particular - we would be in a stronger position to design strategies to disrupt these functional ties and to inhibi parasite infection. However, with the exception of the well-recognized auxotrophies for purines, polyamines and certain amino acids, the metabolic dependencies of intracellular T. cruzi amastigotes have not been described in any cell type. This proposal builds on results from a recent genome-wide RNA interference (RNAi) screen in HeLa cells that offers an unbiased preview of host susceptibility factors that support intracellular T. cruzi infection. Among the pathways identified in our screen, host fatty acid metabolism emerged as a prominent candidate pathway that positively impacts intracellular T. cruzi amastigote growth. Proposed studies seek to determine where and how intracellular T. cruzi amastigotes intersect host fatty acid -oxidation and synthesis pathways and where the critical dependencies for this pathogen lie. Studies will exploit induced pluripotent stem cell-derived (iPSC) human cardiomyocytes as a new and biologically relevant T. cruzi infection model. RNAi will be combined with extracellular metabolic flux analyses and metabolic mass spectrometry to identify specific components (lipid entities and host enzymes) of host fatty acid metabolism that are critical to supporting human cardiomyocyte infection by T. cruzi. The proposed molecular and phenotypic characterization of host fatty acid metabolism as a key regulator of intracellular T. cruzi growth in cardiomyocytes will generate novel insights into the biology of this important human pathogen and its ability to integrate its metabolic needs with the host. Knowledge of these core dependencies has the potential to inform therapeutic strategies aimed at uncoupling the pathogen from its host.